Interaction Screening by Immunoprecipitation

Immunoprecipitation of tagged or untagged proteins and subsequent analysis of interacting proteins by mass spectrometry has led to the construction of organism-wide protein-protein interaction maps, for example in yeast (Gavin et al. 2002, 2006; Ho et al. 2002), and to the identification of novel players even in the well-studied EGF signaling pathway in mammals (Blagoev et al. 2003; Schulze et al. 2005).

The so-called tandem affinity purification tag (TAP-tag) has been developed to obtain highly purified protein complexes (Fig. 2). It consists of a protein A domain that can bind to IgG-agarose followed by a tobacco etch virus (TEV) protease recognition site, which allows the fusion protein to be cleaved from the IgG-agarose. The remaining protein complex is then purified again in a calcium-dependent manner over calmodulin-agarose, making use of the calmodulin binding protein domain (CBP domain). Proteins interacting with the bait protein can be identified by mass spectrometry after elution (Rigaut et al. 1999). For the efficient use of the TAP-tag principle in plants, a synthetic gene has been constructed with optimal codon usage and deletion of a cryptic nuclear localization signal (Rohila et al. 2004). This system was successfully applied to screen for interaction partners of 41 TAP-tagged protein kinases in rice (Rohila et al. 2006). However, full interpretation of this dataset suffers from poor annotation of the rice proteome.

Immunoprecipitation of epitope-tagged BRI1 and BAK1 (tagged either with FLAG tag or GFP) was used in a thorough analysis of receptor phos-phorylation under different ligand concentrations (Wang et al. 2005). In these experiments the analysis of phosphorylation sites of the tagged bait proteins

Fig. 2 Principle of TAP-tag purification. Overview of the experimental workflow using TAP-tagged proteins. The tagged protein is expressed in plants or cells and subsequently purified using IgG-agarose, TEV-protease cleavage, and calmodulin-agarose. Eluted proteins are separated on a one-dimensional SDS gel and individual bands are analyzed by mass spectrometry. C: chloroplast; N: nucleus; V: vacuole

Fig. 2 Principle of TAP-tag purification. Overview of the experimental workflow using TAP-tagged proteins. The tagged protein is expressed in plants or cells and subsequently purified using IgG-agarose, TEV-protease cleavage, and calmodulin-agarose. Eluted proteins are separated on a one-dimensional SDS gel and individual bands are analyzed by mass spectrometry. C: chloroplast; N: nucleus; V: vacuole themselves was the primary aim and not the identification of interaction partners; thus, tandem purification was not applied. This work on brassinosteroid signaling is one of the first clear examples in plants in which a receptor kinase was shown to be (auto)phosphorylated in a ligand-dependent manner.